This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. An understanding of the mechanisms regulating differentiation is clearly relevant to understanding and treatment of cancer and many other diseases, and will be necessary to make tissue differentiation in vitro or from stem cells a reality. Differentiation of tissue types results from differential gene expression, regulated in part at the level of the transcriptome. The homeodomain transcription factor Pax6 regulates eye-specific gene expression in such diverse metazoans as the fruit fly Drosophila melanogaster, mice and humans. Temporal and spatial information provided by signaling pathways is also essential for eye development. However, the transcriptional networks that govern eye development are extremely complex, and traditional genetic approaches alone have not been able to untangle the relationships among the individual factors. What is needed is a complete knowledge of the genes involved and how they interact with one another. Recent advances in massively parallel sequencing have made possible new approaches to transcriptome analysis. A complete understanding of the transcriptional networks that regulate differentiation of the eye will require knowledge of how the intrinsic input from tissue-specific transcription factors like Pax6 is integrated with extrinsic input from signaling pathways. Using the D. melanogaster eye as a model, massively parallel sequencing will be used to identify transcriptional targets potentially co-regulated by Pax6 and by signaling pathways important for eye development. Subsequently, computational approaches will be used to identify genes with similar expression profiles, and to identify genes directly regulated by Pax6 and the nuclear effectors of these signaling pathways.